Diagnostic laboratory tests on patient bodily fluids or tissue specimens provide significant information for disease treatment. These tests aid in discovery of occult disease, early diagnosis after the onset of signs and symptoms, differential diagnosis of various possible diseases, determination of the stage of the disease, estimation of the activity of the disease, detection of recurrence of disease, and measurement of the efficacy of therapy. The diagnostic benefits of many laboratory tests, however, require that the chemical and physical properties of a patient's sample remain substantially unaltered during transport, i.e., the time required to go from sample collection from the patient to processing and/or testing by the laboratory.
Microbial contamination of a patient's sample can cause significant changes to its physical and biochemical composition during transport. If the bioburden of a sample is low and the sample is processed within a short time (2 to 4 hours) after collection, the microorganisms present usually will not affect the sample's chemical properties. However, if the sample can sustain the growth of organisms and the time between collection and processing exceeds 4 hours, the growth and/or metabolism of the microorganisms can alter the chemical and/or physical properties of the sample. For example, the organisms may be able to consume certain components present in the sample such as carbon, nitrogen, or minerals, thus, removing or altering components which were present in the sample at the time of collection. Secondly, as a result of microbial growth, metabolism or death, components which were not present at the time of collection may be released into the sample.
One type of patient sample which is greatly affected by microbial contamination during transport is a urine sample submitted for urinalysis. Urinalysis is a series of tests performed on the urine sample including leukocytes, cast cells, red blood cells, glucose, bilirubin, ketone, specific gravity, pH, protein, urobilinogen, nitrite, and blood. Normal healthy women have 102-103 microorganisms present in their urine. Microorganisms can also be introduced into the patient's urine sample either through a patient's clinical infection or by inadvertent contamination during the collection process or transport to the laboratory. Since urine samples generally contain sufficient metabolites and other factors required to support the growth and replication of most microorganisms commonly found in urine samples, delays in transport beyond 4 hours can lead to significant changes in the chemical and physical properties of the samples. For example, in one study, common urinary tract contaminants or potential urinary pathogens significantly altered the chemical/physical properties of unpreserved urine held for 8-24 hours or longer at room temperature: false positive reactions for hemoglobin, protein, nitrites, and esterase; false negative reactions for nitrites, glucose, protein and ketones; and substantial changes in pH. Many of these alterations in the chemical properties of the unpreserved urine samples occurred within 8 hours (Dorn, G. L., unpublished).
Laboratory standards recommend that urine samples be analyzed within two hours after collection from the patient to circumvent changes in chemical and physical properties. However, with the emergence of Health Maintenance Organizations (HMO's), Preferred Physician Organizations (PPO's), and centralized laboratory testing facilities combined with increasing pressure to be more cost effective through batch processing of samples, it has become increasingly difficult to comply with traditional standards of practice. For example, one study showed that a large percentage of samples submitted for culture within a hospital setting were>4 hours old prior to processing. (Dorn, et al., “Adherence to laboratory guidelines: a study on urine specimen transport time,” Diagnostics & Clinical Testing 27:28-31 (1989)). Another study indicated that all samples submitted to centralized commercial laboratories exceeded the recommended time limits for transport. (Dorn, G. L., “Microbial stabilization of antibiotic-containing urine samples by using the FLORASTAT urine transport system,” J Clin Micro 29:2169-2174 (1991)). In the area of urinalysis, a recent College of American Pathologist Q-Probes Study documented that for inpatients and outpatients, respectively, only 64% and 77% of laboratories were able to meet the 2-hour transport goal 90% of the time. (Howanitz, et al., “Timeliness of urinalysis: a College of American Pathologists Q-probes study of 346 small hospitals,” Arch Pathol Lab Med 121:667-672 (1997)).
Since current medical practices often prevent expedient transport of urine samples for urinalysis, and since microorganisms are frequently present in urine samples collected for urinalysis, a method of blocking the deleterious effects of microbial contamination on a urine sample during transport is advantageous in preserving the chemical and physical integrity of the sample. For this purpose, various preservatives for urine samples have been developed and commercialized. Among the active ingredients for these preservatives are: boric acid, mercuric oxide, sodium azide, tartaric acid, and thimerosal (ethylmercurithiosalicylic acid). Boric acid has been reported to be compatible as a preservative used in combination with urinalysis and leukocyte/cast cell analysis. (Porter, I. A. and Brodie, J. 1969. “Boric acid preservation of urine samples,” Br Med J 2:353-355; Guenther, K. L. and Washington II, J. A. 1981. Evaluation of the B-D urine culture kit,” J Clin Microbiol 14:628-630).
Despite the commercial availability of preservatives for urine samples, there are significant deficiencies associated with them. For example, many of the active ingredients used in commercially available urine preservative systems present health, flammability, and/or reactivity hazards. According to the National Fire Prevention Association's health, flammability, and reactivity hazard ratings for chemicals, a short exposure to mercuric oxide and thimerosal could cause death or major residual injury. Although mercury-based systems such as mercuric oxide (Starplex Scientific, Inc., Etobicoke, Ontario, Canada) and thimerosal (Sigma Chemical Co., St. Louis, Mo.) effectively stabilize samples, their high National Fire Protection Association (NFPA) health hazard rating makes them unsuitable for use when large numbers of samples or high volumes of material are being processed. While sodium azide (Sigma Chemical Co.) is also an effective stabilizing material, the health rating assigned to sodium azide indicates that short exposure could cause serious temporary or residual health injury, making it unsuitable for use in high volume processing. While boric acid (Becton Dickinson, Franklin Lakes, N.J.) (Sage, Inc., Crystal Lakes, Ill.) (Bibby Sterlin, Ltd., Dynalab Corp., Rochester, N.Y.) and tartaric acid (Mid-America Health, Niagara Falls, N.Y.) have moderate to low NFPA hazard ratings, they are not cidal and, consequently, do not effectively block the deleterious effects of all microorganisms of interest, potentially causing false negative and false positive urinalysis results when the urine sample is held at room temperature beyond 8 hours. Therefore, as illustrated in the case of urine samples submitted for urinalysis, there is a continuing need for an effective transport system which provides stability of the chemical and physical properties of patient specimens and bodily fluids without exposing patients, healthcare professionals, and laboratory personnel to serious health hazards.
Biological reagents, some of which are often used in diagnostic testing procedures, are also susceptible to chemical and physical alteration due to microbial contamination. These reagents contain substances which are critical to their function but also capable of supporting microbial growth and/or metabolism. Although most biological reagents are manufactured under sterile conditions in sealed containers, low level microbial contamination can occur during manufacturing. During storage, the growth of the contaminating microorganisms can cause chemical and physical changes to the reagent. Moreover, many reagents are sold in multiple-entry containers at volumes which allow the user to repetitively extract small aliquots over time. There exists the possibility of microbial contamination of the reagent at each entry event. One commercially available preservative for reagents is Micr-O-protect™ (Roche Diagnostics, GmbH, Mannheim, Germany), an ethanolic solution of bromonitrodioxane and methylisothiazolone, with a health rating indicating that short exposure could cause serious temporary or residual injury and a flammability rating indicating that it could be ignited under most ambient conditions. Another preservative is the StabilZyme Select® Conjugate Stabilizer (SurModics, Inc., Eden Prairie, Minn.) which is an aqueous protein-containing mixture preserved with methylisothiazolone and bromonitrodioxane. Yet another line of preservative is ProClin (Supelco Inc., Bellefonte, Pa.) which utilizes 5-chloro-2-methyl-4-isothiazolin3-one and 2-methyl-4-isothiazolin-3-one. Isothiazolone and its derivatives are corrosive to the eyes potentially causing permanent irreversible injury, can cause skin burns or irritation, and are considered toxic to fish and wildlife if permitted to enter the water supply. Bromonitrodioxane is a formaldehyde releaser, and since formaldehyde is carcinogenic and highly flammable in liquid and gaseous forms, bromonitrodioxane is an unfavorable candidate as a preservative for samples processed in high volume. Consequently, there is a need for an environmentally friendly system which can preserve a biological reagent while maintaining its chemical and physical properties.
Preservatives are often added to therapeutics to increase shelf-life and to reduce the possibility of microbial contamination. As in the case of biological reagents, many therapeutics are packaged in multiple-entry containers at volumes which allow the extraction of small aliquots over time. For example, vaccines are routinely provided in multiple entry containers, and for several decades, thimerosal, a mercury-based preservative, has been used in vaccines to prevent contamination and other biologics in multidose containers. The Food and Drug Administration (FDA) has undertaken a review of drugs containing mercury-based preservatives, including thimerosal, in an effort to reduce the concentration of mercury in vaccines and to find alternative preservative formulations that do not contain mercury. (“Recommendations regarding the use of vaccines that contain thimerosal as a preservative,” MMWR 48:996-998 (Nov. 5, 1999)) Therefore, there is a need to provide safe, effective preservatives for therapeutics which reduce the risk of microbial contamination as well as potential health problems associated with exposure to mercury.
Microbial contamination can also lead to the chemical and/or physical degradation of personal care products such as cosmetics, hand cleansers, lotions, and shampoos. Moreover, contaminated products routinely exhibit diminished performance and contribute to the spread of infection to users.
Work surfaces and equipment in hospitals and laboratories are highly susceptible to microbial contamination. Likewise, surfaces and appliances found in kitchens and bathrooms of households, restaurants, groceries, catering establishments and the like are routinely exposed to microbial contamination. There is a continuing need for environmentally safe, effective sanitizing products capable of reducing the microbial bioburden in these areas as well.